Crude drug composition for preventing or treating respiratory diseases

ABSTRACT

The present disclosure relates to a pharmaceutical composition for preventing or treating respiratory diseases and a food composition for preventing or relieving respiratory diseases comprising a mixed extract of Dioscoreae rhizoma, Taraxaci herba and Schizonepetae spica as an active ingredient. A composition comprising a mixed extract of Dioscoreae rhizoma, Taraxaci herba and Schizonepetae spica of the present disclosure has an advantage of preventing respiratory diseases and relieving the symptoms of respiratory diseases, restores damages in the lung tissue and has efficacy for inhibiting inflammation, oxidative stress and ageing reactions induced by fine dust. More particularly, the composition of the present disclosure increases the expression of a telomerase which extends the length of a telomere, and thus is expected to fundamentally treat degenerative respiratory diseases induced by fine dust unlike existing symptom alleviating agent.

FIELD

The present application claims priorities from Korean Patent ApplicationNo. 10-2018-0031150 filed with the Korean Intellectual Property Officeon 16 Mar. 2018 and Korean Patent Application No. 10-2019-0028956 filedwith the Korean Intellectual Property Office on 13 Mar. 2019, thedisclosures of which are incorporated herein by reference.

The present disclosure relates to an herbal medicine composition forprevention or treatment of a respiratory disease and, specifically, to acomposition for prevention, treatment, or alleviation of a respiratorydisease, the composition comprising extracts of Dioscoreae rhizoma,Taraxaci herba, and Schizonepetae spica.

BACKGROUND

Respiratory diseases are diseases in connection with the lungs andairways, and may be mainly caused by lowered immunity, inflammatoryactions, bacterial or viral infections, inhalation of harmful particlesdue to fine dust or smoking, aging, and the like. Representativerespiratory diseases include pneumonia, rhinitis, asthma, bronchitis,tuberculosis, chronic obstructive pulmonary disease (COPD), and thelike. Particularly, chronic obstructive pulmonary disease patients arerecently increasing due to an increase in fine dust, smoking, and thelike. Chronic obstructive pulmonary disease is also called emphysema orchronic bronchitis.

Medicines for such respiratory diseases are being developed mainlytargeting anti-inflammatory actions or airway dilation effects. Examplesof respiratory disease medicines showing anti-inflammatory and airwaydilation effects are glucocorticoid steroid drugs, beta₂-adrenergicreceptor agonists, leukotriene receptor antagonists, andphosphodiesterase-4 inhibitors (PDE4 inhibitors). However, thetherapeutic purposes of these existing respiratory disease medicines arerestricted to allergic asthma in infants or children and chronicobstructive pulmonary disease (COPD) in smokers. Moreover, most of themedicines are used for only a purpose of relieving symptoms, and have alimitation in that the medicines fail to delay or stop the progressionof respiratory diseases through the removal of fundamental causes of therespiratory diseases. Since most of respiratory diseases havecomplicated causes and symptoms, existing medicines using a singlecomponent or a single therapeutic mechanism cannot obtain suitabletherapy. Accordingly, there is an urgent need to develop a novelmedicine for preventing and treating a respiratory disease morediversely and complexly.

SUMMARY Technical Problem

The present inventors have made research efforts for developing arespiratory disease medicine having a novel therapeutic mechanism toovercome the limitations of the existing respiratory disease medicinesdescribed above. As a result, the present inventors verified that acomplex herbal medicine extract (mixed extract) of Dioscoreae rhizoma,Taraxaci herba, and Schizonepetae spica has effects of inhibitinginflammation, oxidative stress, and aging caused by fine dust, as wellas lung tissue damage, and thus have completed the present invention.

Accordingly, an aspect of the present disclosure is to provide apharmaceutical composition for prevention or treatment of a respiratorydisease, the pharmaceutical composition comprising: as an activeingredient, a mixed extract of Dioscoreae rhizoma, Taraxaci herba, andSchizonepetae spica; and a pharmaceutically acceptable carrier.

Another aspect of the present disclosure is to provide a foodcomposition for prevention or alleviation of a respiratory disease, thefood composition comprising, as an active ingredient, a mixed extract ofDioscoreae rhizoma, Taraxaci herba, and Schizonepetae spica.

Technical Solution

In accordance with an aspect of the present disclosure, there isprovided a pharmaceutical composition for prevention or treatment of arespiratory disease, the pharmaceutical composition comprising: (a) asan active ingredient, a mixed extract of Dioscoreae rhizoma, Taraxaciherba, and Schizonepetae spica; and (b) a pharmaceutically acceptablecarrier.

Herein, Dioscoreae rhizoma is a rhizome (stalk with roots) steamed anddried as it is after the main bark of Dioscorea batatas Decaisne orDioscorea japonica Thunberg is removed. The raw material of thismedicine originates in China, but is also grown naturally or cultured inKorea, Japan, and Taiwan. Dioscoreae rhizoma is a cylindrical or unevencylindrical rhizome. In general, the rhizomes are collected in autumn,peeled, and then dried in the shade before use.

Herein, Taraxaci herba indicates a whole plant of Taraxacum platycarpumH. Dahlstedt, Taraxacum officinale Weber, Taraxacum mongolicumHandel-Mazzetti, or Taraxacum coreanum Nakai. Donguibogam describes thatTaraxaci herba shows good effects of releasing heat poisoning,suppressing pestilent lesions, loosening lumps, removing dietarypoisoning, and eliminating indigestion.

Herein, Schizonepetae spica is a floral axis (flower petal) ofSchizonepetae tenuifolia Briquet. Schizonepetae spica is a flower petalthat has a thin and long barley ear shape, is 5-10 cm in length, and ispurple-greenish brown to greenish brown. Small lip-like flowers andoften calyx tubes having fruits are linked thereto. Small milky shortfurs can be also seen on the entirety of the stalks. Schizonepetae spicais known to have warmth, non-toxicity, and spiciness.

The extracts of Dioscoreae rhizoma, Taraxaci herba, and Schizonepetaespica used in the present disclosure may be purchased, or obtained bydirect extraction from the herbal medicines. The extraction may beperformed after the respective herbal medicines are cut or pulverizedinto proper sizes.

In a case where the extracts used in the composition of the presentdisclosure are obtained by direct extraction from the herbal medicinesDioscoreae rhizoma, Taraxaci herba, and Schizonepetae spica, variousextraction solvents, such as polar solvents or non-polar solvents, maybe used.

Suitable polar solvents may include (i) water, (ii) a C1 to C6 loweralcohol (specifically, methanol, ethanol, propanol, butanol, n-propanol,iso-propanol, n-butanol, 1-pentanol, 2-butoxyethanol, or ethyleneglycol), (iii) acetic acid, (iv) dimethyl-formamide (DMFO), and (v)dimethyl sulfoxide (DMSO). Suitable non-polar solvents include acetone,acetonitrile, ethyl acetate, methyl acetate, fluoroalkanes, pentane,hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane,diisobutylene, 1-pentene, 1-chlorobutane, 1-chloropentane, o-xylene,diisopropylether, 2-chloropropane, toluene, 1-chloropropane,chlorobenzene, benzene, diethyl ether, diethyl sulfide, chloroform,dichloromethane, 1,2-dichloroethane, aniline, diethyl amine, ether,carbon tetrachloride, and tetrahydrofuran (THF).

The amount of the extraction solvent may vary depending on the amountsof Dioscoreae rhizoma, Taraxaci herba, and Schizonepetae spica to besubjected to extraction, and specifically, the extraction solvent has avolume 1-20 times, specifically, 5-15 times, more specifically, 5-12times, or 7-12 times the weight of Dioscoreae rhizoma, Taraxaci herba,and Schizonepetae spica, or a mixture thereof. Most specifically, theextraction solvent has a volume 10 times the weight of Dioscoreaerhizoma, Taraxaci herba, Schizonepetae spica, or a mixture thereof.

The extraction temperature of the extracts of the present disclosure isnot particularly limited, and the extraction temperature may be forexample 0-120° C., and specifically, 15-95° C. In an embodiment of thepresent disclosure, the extraction temperature is room temperature.

The extraction time of the extracts of the present disclosure is notparticularly limited, and the extraction time may be for example 1 hourto 10 days, specifically, 1-72 hours, 1-48 hours, 1-36 hours, 1-24hours, 1-12 hours, 1-10 hours, or 1-6 hours. The extraction time may bemore specifically, 2-72 hours, 2-48 hours, 2-36 hours, 2-24 hours, 2-12hours, 2-10 hours, 2-6 hours, 3-72 hours, 3-48 hours, 3-36 hours, 3-24hours, 3-12 hours, 3-10 hours, 5-48 hours, 5-36 hours, 5-24 hours, 5-12hours, 5-10 hours, 6-12 hours, or 6-10 hours, and most specifically, 8hours.

The extracts of the present disclosure may be extracted by a knownnatural substance extraction method. For example, the extraction may becarried out by cold extraction, hot-water extraction, ultrasonicextraction, reflux cooling extraction, or heating extraction, andspecifically, cold extraction. The extraction may be repeated one to tentimes, and more specifically two to seven times.

According to an embodiment of the present disclosure, the extracts ofDioscoreae rhizoma, Taraxaci herba, and Schizonepetae spica used in thepresent disclosure can be extracted with an organic solvent, water, or amixed solvent thereof. Examples of the organic solvent include a C1 toC6 lower alcohol, petroleum ether, hexane, benzene, chloroform,methylene chloride, ether, ethyl acetate, and acetone.

The concentration of the organic solvents, such as a C1 to C6 loweralcohol, petroleum ether, hexane, benzene, chloroform, methylenechloride, ether, ethyl acetate, and acetone, may be 1-100% (v/v),specifically 10-100% (w/w), 20-100% (w/w), 30-100% (w/w), 40-100% (w/w),50-100% (w/w), 60-100% (w/w), 70-100% (w/w), 80-100% (w/w), and morespecifically 10-90% (w/w), 10-80% (w/w), 10-70% (w/w), 10-60% (w/w),10-50% (w/w), 10-40% (w/w), or 10-30% (w/w), still more specifically20-80% (w/w), 20-70% (w/w), 20-60% (w/w), 20-50% (w/w), 20-40% (w/w), or20-30% (w/w), and most specifically 25% (w/w), but is not limitedthereto.

According to still another embodiment of the present disclosure, theextracts of Dioscoreae rhizoma, Taraxaci herba, and Schizonepetae spicaof the present disclosure may be extracted with water, a C1 to C6 loweralcohol, or a mixed solvent thereof as described above, or may be, afterextraction and concentration (under reduced pressure), further extractedor fractionated with an organic solvent selected from the groupconsisting of petroleum ether, hexane, benzene, chloroform, methylenechloride, ether, ethyl acetate, and acetone as described above.

Meanwhile, the mixed extract of the extracts of Dioscoreae rhizoma,Taraxaci herba, and Schizonepetae spica used in the present disclosuremay be prepared by mixing individual extracts of Dioscoreae rhizoma,Taraxaci herba, and Schizonepetae spica, or may be prepared by treatinga mixture of the herbal medicines Dioscoreae rhizoma, Taraxaci herba,and Schizonepetae spica with an extraction solvent.

In the present disclosure, the extracts of Dioscoreae rhizoma, Taraxaciherba, and Schizonepetae spica may be used in the form of a crudeextract extracted by a solvent, and may be used through high-puritypurification.

As used herein, the term “extract” has a meaning that is commonly usedas a crude extract in the art as described above, and broadly,encompasses a fraction obtained by additionally fractionating theextract. In other words, the extracts of Dioscoreae rhizoma, Taraxaciherba, and Schizonepetae spica include not only ones obtained by usingthe above-described extraction solvents but also ones obtained byadditionally applying a purification procedure to the same. For example,the extracts of Dioscoreae rhizoma, Taraxaci herba, and Schizonepetaespica of the present disclosure include fractions obtained throughvarious purification methods that are additionally performed, such as afraction obtained by passing the extracts through an ultrafiltrationmembrane with a predetermined molecular weight cut-off value and afraction obtained by various types of chromatography (fabricated forseparation depending on size, charge, hydrophobicity, orhydrophilicity). The extracts of Dioscoreae rhizoma, Taraxaci herba, andSchizonepetae spica used in the present disclosure may be prepared in apowder type by additional procedures, such as distillation under reducedpressure and freeze-drying or spray drying.

According to a specific embodiment of the present disclosure, Dioscoreaerhizoma, Taraxaci herba, and Schizonepetae spica are washed and dried,and then mixed at a predetermined weight ratio. Thereafter, the mixtureis placed in an extraction solvent having a volume (ml) 1-20 times theweight (g) thereof, and then subjected to extraction while well stirredat 15-95° C. for 1-48 hours. Then, the extract was filtered,concentrated under reduced pressure at 50-65° C., and then freeze-dried,thereby obtaining a powder-type complex herbal medicine extract (mixedextract).

According to another embodiment of the present disclosure, Dioscoreaerhizoma, Taraxaci herba, and Schizonepetae spica are separately placedin an extraction solvent having a volume (ml) 1-20 times the weight (g)thereof, and then subjected to extraction while well stirred at 15-95°C. for 1-48 hours. Then, each extract is filtered, concentrated underreduced pressure at 50-65° C., and then freeze-dried, thereby obtaininga powder-type herbal medicine extract. Thereafter, the respective herbalmedicine extracts are mixed at a predetermined weight ratio, therebyobtaining a powder-type complex herbal medicine extract (mixed extract).

According to a particular embodiment of the present disclosure,Dioscoreae rhizoma, Taraxaci herba, and Schizonepetae spica are mixed ata weight ratio of 1:1:1, and then ethanol having a volume 10 times theweight is added thereto, followed by extraction at room temperature for8 hours. The extract is concentrated under reduced pressure at 50-65°C., and then the concentrate is freeze-dried, thereby obtaining acomplex herbal medicine extract of Dioscoreae rhizoma, Taraxaci herba,and Schizonepetae spica.

The mixed extract of three kinds, Dioscoreae rhizoma, Taraxaci herba,and Schizonepetae spica used in the present disclosure may containherbal medicine extracts of Dioscoreae rhizoma, Taraxaci herba, andSchizonepetae spica at a mixing weight ratio of 1-10:1-10:1-10,1:1-10:1-10, 1-10:1:1-10, 1-10:1-10:1, 1:1:1-10, 1:1-10:1, or 1-10:1:1,at a mixing weight ratio of 1-5:1-5:1-5, 1:1-5:1-5, 1-5:1:1-5,1-5:1-5:1, 1:1:1-5, 1:1-5:1, or 1-5:1:1, at a mixing weight ratio of1-4:1-4:1-4, 1:1-4:1-4, 1-4:1:1-4, 1-4:1-4:1, 1:1:1-4, 1:1-4:1, or1-4:1:1, or at a mixing weight ratio of 1-3:1-3:1-3, 1:1-3:1-3,1-3:1:1-3, 1-3:1-3:1, 1:1:1-3, 1:1-3:1, 1-3:1:1, or 1:1:1.

The mixing ratio of the herbal medicine ingredients described herein iscalculated on the basis of the weight of solvent-removed solids (in acase of a mixture of herbal medicine extracts) or the weight of herbalmedicines per se (in a case of an extract of mixed herbal medicines).

As used herein, the term “to” or “-” between two numerical values refersto an interval between the numerical values including numerical valuesdescribed before and after the term.

The composition of the present disclosure may be prepared into apharmaceutical composition.

According to a specific embodiment of the present disclosure, thecomposition of the present disclosure is a pharmaceutical compositioncomprising: (a) as an active ingredient, the above-described mixedextract of Dioscoreae rhizoma, Taraxaci herba, and Schizonepetae spica;and (b) a pharmaceutically acceptable carrier.

As used herein, the term “pharmaceutical effective amount” refers to anamount sufficient to attain efficacy of the above-described mixedextract of Dioscoreae rhizoma, Taraxaci herba, and Schizonepetae spicato treat or prevent a respiratory disease. The present disclosure isdirected to a composition comprising extracts extracted from the naturalplant materials, Dioscoreae rhizoma, Taraxaci herba, and Schizonepetaespica, and even the administration of an excess of the compositioncauses no side effects in the human body, and thus a person skilled inthe art could select and implement the upper limit of the amount of theextracts contained in the composition of the present disclosure.

As used herein, the term “respiratory disease” includes a respiratorydisease selected from the group consisting of a cold, rhinitis,pharyngitis, laryngitis, pharyngolaryngitis, acute or chronic pneumonia,acute or chronic bronchitis, asthma, and chronic obstructive pulmonarydisease, but is not limited thereto.

As validated in the following examples, the administration of thecomplex herbal medicine extract (mixed extract) of Dioscoreae rhizoma,Taraxaci herba, and Schizonepetae spica shows an effect of significantlyreducing lung tissue damage in emphysema mouse models.

In addition, the complex herbal medicine extract of the presentdisclosure significantly inhibits reactive oxygen species generation,apoptosis, and aging in pulmonary epithelial cell lines, induced by finedust, and dose-dependently increases the expression degree of telomerasethat extends the length of telomere.

In addition, the complex herbal medicine extract of the presentdisclosure shows an excellent anti-inflammatory effect by inhibiting NOproduction of the mouse macrophage cell line treated with LPS.

The complex herbal medicine extract of the present disclosure also showsan excellent anti-inflammatory effect by inhibiting the expression ofthe inflammatory factors (IL-6, IL-1β, and iNOS) expressed in the mousemacrophage cell line treated with LPS.

The complex herbal medicine extract of the present disclosure also showsan excellent antioxidative effect by increasing the expression of theantioxidative factor HO-1 in the mouse macrophage cell line treated withLPS, and shows an excellent antioxidative effect even in various ratioranges of herbal medicines even when the cell lines were treated withcomplex herbal medicine extracts comprising herbal medicines at variousratios.

The complex herbal medicine extract of the present disclosure also showsan excellent anti-inflammatory effect by inhibiting the expression of ananti-inflammatory factor (iNOS) even when the extract is extracted withan extraction solvent (ethanol) at various concentrations, and shows anexcellent antioxidative effect by increasing the expression of theantioxidative factor HO-1 even when the concentrations and kinds of theextraction solvent are different.

As well, the complex herbal medicine extract of the present disclosure,compared with a single herbal medicine extract, significantly inhibitsthe expression of inflammatory factors (IL-6, IL-1β, and TNF-α) in thelung tissue of the pulmonary inflammation models induced by LPS, therebyshowing a synergistic effect in an anti-inflammatory effect comparedwith a single herbal medicine extract.

The above results of the examples indicate that the compositioncomprising the complex herbal medicine extract of the present disclosurecan be used in the prevention, alleviation, or treatment of arespiratory disease, such as pneumonia, bronchitis, chronic obstructivepulmonary disease, or asthma, caused by inflammation and aging, and canbe used as an alternative for a conventional respiratory diseasemedicine.

In cases where the composition of the present disclosure is preparedinto a pharmaceutical composition, the pharmaceutical composition of thepresent disclosure may contain a pharmaceutically acceptable carrier.The pharmaceutically acceptable carrier is normally used at the time offormulation, and examples thereof may include, but are not limited to,lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum,calcium phosphate, alginate, gelatin, calcium silicate, microcrystallinecellulose, polyvinylpyrrolidone, cellulose, water, syrup,methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc,magnesium stearate, mineral oil, and the like. The pharmaceuticalcomposition of the present disclosure may further contain, in additionto the above ingredients, a lubricant, a humectant, a sweetener, aflavoring agent, an emulsifier, a suspending agent, a preservative, andthe like. Suitable pharmaceutically acceptable carriers and preparationsare described in detail in Remington's Pharmaceutical Sciences (19thed., 1995).

The pharmaceutical composition of the present disclosure may beadministered orally or parenterally, and examples of parenteraladministration may include intravenous administration, subcutaneousadministration, intradermal administration, intramuscularadministration, intranasal administration, mucosal administration,intradural administration, intraperitoneal administration, intraocularadministration, and the like, and specifically, the pharmaceuticalcomposition of the present disclosure may be administered orally.

The suitable dose of the pharmaceutical composition of the presentdisclosure varies depending on factors, such as a formulating method, amanner of administration, patient's age, body weight, gender, morbidity,and food, a time of administration, a route of administration, anexcretion rate, and response sensitivity. The ordinarily skilledpractitioners can easily determine and prescribe the dose that iseffective for the desired treatment or prevention. According to aspecific embodiment of the present disclosure, the daily dose of thepharmaceutical composition of the present disclosure is 0.001-1,000mg/kg. The daily dose of the pharmaceutical composition of the presentdisclosure may be for example 0.1-1000 mg/kg, 0.1-900 mg/kg, 0.1-800mg/kg, 0.1-700 mg/kg, 0.1-600 mg/kg, 0.1-500 mg/kg, 0.1-400 mg/kg,0.1-300 mg/kg, 0.1-200 mg/kg, 0.1-100 mg/kg, 0.1-50 mg/kg, 0.1-30 mg/kg,0.1-20 mg/kg, 0.1-10 mg/kg, 0.1-7 mg/kg, or 0.1-5 mg/kg, and may be1-1000 mg/kg, 1-900 mg/kg, 1-800 mg/kg, 1-700 mg/kg, 1-600 mg/kg, 1-500mg/kg, 1-400 mg/kg, 1-300 mg/kg, 1-200 mg/kg, 1-100 mg/kg, 1-50 mg/kg,1-30 mg/kg, 1-20 mg/kg, 1-10 mg/kg, 1-7 mg/kg, or 1-5 mg/kg, and morespecifically may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg. In anotherembodiment, the daily dose of the pharmaceutical composition of thepresent disclosure may be for example 100-900 mg/kg, 100-800 mg/kg,100-700 mg/kg, 100-600 mg/kg, 100-500 mg/kg, 100-400 mg/kg, 100-300mg/kg, or 100-200 mg/kg, may be 200-900 mg/kg, 200-800 mg/kg, 200-700mg/kg, 200-600 mg/kg, 200-500 mg/kg, 200-400 mg/kg, or 200-300 mg/kg,may be 300-900 mg/kg, 300-800 mg/kg, 300-700 mg/kg, 300-600 mg/kg,300-500 mg/kg, or 300-400 mg/kg, and may be 400-900 mg/kg, 400-800mg/kg, 400-700 mg/kg, 400-600 mg/kg, or 400-500 mg/kg, and morespecifically, 100 mg/kg, 200 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, or 1000 mg/kg, but is notlimited thereto.

The pharmaceutical composition of the present disclosure may beformulated into a unit dosage form or may be prepared in a multi-dosecontainer by using a pharmaceutically acceptable carrier and/orexcipient according to a method that can be easily implemented by aperson having an ordinary skill in the art to which the presentdisclosure pertains. The formulation may be a solution in an oily oraqueous medium, a suspension, an emulsion, an extract, a powder,granules, a tablet, or a capsule, and may further contain a dispersantor a stabilizer.

The pharmaceutical composition of the present disclosure may beadministered in combination with a known compound or pharmaceuticalcomposition having effects of preventing and treating a respiratorydisease or respiratory disease-related symptoms.

According to another aspect of the present disclosure, there is provideda food composition for prevention or alleviation of a respiratorydisease, the food composition comprising, as an active ingredient, amixed extract of Dioscoreae rhizoma, Taraxaci herba, and Schizonepetaespica. The food composition may be used as a health functional food ormay be added to various types of foods.

The present disclosure also provides a health functional food comprisingthe food composition. The health functional food may be drinks, meats,chocolates, foods, confectionery, pizzas, instant noodles, othernoodles, gums, ice creams, alcohol drinks, vitamin complexes, and healthsupplement foods.

The content of the mixed extract of the present disclosure contained inthe food composition may be appropriately controlled according to theform of food, the desired use, or the like, and is not particularlylimited thereto. For example, the content of the mixed extract may be0.001-30 wt % or 0.01-20 wt % of the entire food weight, and the healthfood composition may be 0.001-15 g, 0.02-10 g, or 0.3-1 g on the basisof 100 ml thereof, but is not limited thereto.

The composition comprising a mixed extract of Dioscoreae rhizoma,Taraxaci herba, and Schizonepetae spica of the present disclosure, whenprepared into a food composition, may contain ingredients that areordinarily added in the manufacture of foods as well as the extract asan active ingredient. The added ingredients include, for example, aprotein, a carbohydrate, a fat, a nutrient, a seasoning, and a flavoringagent. Examples of the foregoing carbohydrate may include typical sugars(monosaccharides, such as glucose and fructose; disaccharides, such asmaltose, sucrose, and oligosaccharides; and polysaccharides, such asdextrin and cyclodextrin) and sugar alcohols, such as xylitol, sorbitol,and erythritol. Examples of the flavoring agent may include naturalflavoring agents (thaumatin, and stevia extract (e.g., rebaudioside A,glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin,aspartame, etc.). For example, the food composition of the presentdisclosure, when is prepared into a drink, may further contain citricacid, liquefied fructose, sugar, glucose, acetic acid, malic acid, fruitjuice, an Eucommia ulmoides extract, a jujube extract, and a licoriceextract, in addition to the extract of the present disclosure.

Since the food composition for prevention or alleviation of arespiratory disease of the present disclosure contains, as an activeingredient, a mixed extract of Dioscoreae rhizoma, Taraxaci herba, andSchizonepetae spica in the same manner as “the pharmaceuticalcomposition for prevention or treatment of a respiratory disease” asdescribed above, a description of overlapping contents therebetween isomitted to avoid excessive redundancy of the present specification.

According to still another aspect of the present disclosure, there isprovided a method for prevention or treatment of a respiratory disease,the method including administering, to a subject, the pharmaceuticalcomposition comprising, as an active ingredient, the above-describedmixed extract of Dioscoreae rhizoma, Taraxaci herba, and Schizonepetaespica of the present disclosure.

According to another aspect of the present disclosure, there is provideda method for prevention or alleviation of a respiratory disease, themethod including administering, to a subject, the food compositioncomprising, as an active ingredient, the above-described mixed extractof Dioscoreae rhizoma, Taraxaci herba, and Schizonepetae spica of thepresent disclosure.

The respiratory disease, which is the target disease of the treatmentmethod or alleviation method of the present disclosure, is the same asdefined in relation to the respiratory disease, which is the targetdisease to be treated with the pharmaceutical composition.

As used herein, the term “administration” or “administer” refers to thedirect administration of a therapeutically or amelioratively effectiveamount of the composition of the present disclosure to a subject (i.e.,an individual) undergoing a respiratory disease, thereby forming thesame amount thereof in the body of the subject.

The term “therapeutically effective amount” of the composition refers tothe content of the composition, which is sufficient to provide atherapeutic or preventive effect to a subject to which composition isadministered, and thus the term has a meaning encompassing“prophylactically effective amount.” As used herein, the term “subject”is a mammal including a human, a mouse, a rat, a guinea pig, a dog, acat, a horse, a cow, a pig, a monkey, a chimpanzee, a baboon, or arhesus monkey. Most specifically, the subject of the present disclosureis a human.

Since the method for prevention, alleviation, or treatment of arespiratory disease of the present disclosure includes administering thepharmaceutical composition for prevention or treatment or the foodcomposition for prevention or alleviation of a respiratory diseaseaccording to an aspect of the present disclosure, a description ofoverlapping contents therebetween is omitted to avoid excessiveredundancy of the present specification.

Advantageous Effects

Features and advantages of the present disclosure are summarized asfollows.

(a) The present disclosure relates to a pharmaceutical composition forprevention or treatment of a respiratory disease and a food compositionfor prevention or alleviation of a respiratory disease, each of thecompositions comprising a mixed extract of Dioscoreae rhizoma, Taraxaciherba, and Schizonepetae spica.

(b) The composition comprising a mixed extract of Dioscoreae rhizoma,Taraxaci herba, and Schizonepetae spica of the present disclosure haseffects of preventing a respiratory disease and relieving symptomsthereof, and has effects of restoring lung tissue damage and inhibitinginflammation, oxidative stress, and aging induced by fine dust.Especially, the composition of the present disclosure is expected to beable to fundamentally treat a degenerative respiratory disease inducedby fine dust, unlike a conventional symptom reliever, by increasing theexpression of telomerase that extends the length of telomeres.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the microscopic observation of lung tissues of PPE-inducedemphysema mouse models, in order to investigate the lung tissue damageinhibitory effect of the complex herbal medicine extract of the presentdisclosure.

FIG. 2 is a graph comparing the degrees of reactive oxygen species (ROS)formation in the pulmonary endothelial cell line, induced by fine dust,in order to investigate the ROS formation inhibitory effect of thecomplex herbal medicine extract of the present disclosure.

FIG. 3 is a graph comparing cell viability according to theconcentrations (100, 200, 400, and 800 μg/ml) of the complex herbalmedicine extract in the pulmonary endothelial cell line treated withfine dust, in order to investigate the apoptosis inhibitory effect ofthe complex herbal medicine extract of the present disclosure.

FIG. 4 shows the results of senescence beta-galactosidase staining afterthe treatment of the pulmonary endothelial cell line with fine dust, inorder to investigate the anti-aging effect of the complex herbalmedicine extract of the present disclosure.

FIG. 5 shows the western blot results of comparing the expressiondegrees of telomerase according to the concentrations (100 and 200μg/ml) of the complex herbal medicine extract in pulmonary endothelialcell lines, in order to investigate the anti-aging effect of the complexherbal medicine extract of the present disclosure.

FIG. 6 is a graph comparing the degrees of nitric oxide (NO) productionaccording to the treatment concentrations (0.5, 1, 2, 4 mg/ml) of thecomplex herbal medicine extract in mouse macrophage cell line treatedwith LPS, in order to investigate the anti-inflammatory effect of thecomplex herbal medicine extract of the present disclosure.

FIGS. 7A, 7B and 7C are graphs showing the expression inhibitory effectof the complex herbal medicine extract of the present disclosure on theexpression of the inflammatory factors (IL-6, IL-113, and iNOS) in mousemacrophage cell line treated with LPS, in order to investigate theanti-inflammatory effect of the complex herbal medicine extract of thepresent disclosure.

FIG. 8 is a graph comparing the expression levels of the antioxidativefactor HO-1 according to the treatment concentrations (0, 0.5, 1, 2mg/ml) of the complex herbal medicine extract in mouse macrophage cellline treated with LPS, in order to investigate the antioxidative effectof the complex herbal medicine extract of the present disclosure.

FIG. 9 is a graph comparing the expression levels of the antioxidativefactor HO-1 according to various ratios of herbal medicines in mousemacrophage cell line treated with LPS, in order to investigate theantioxidative effect according to the mixing ratio in the complex herbalmedicine extract of the present disclosure.

FIG. 10 illustrates graphs comparing the expression levels of theinflammatory factor (iNOS) and the expression levels of theantioxidative factor HO-1 according to the extraction method (hot-waterextraction, 0%) and the concentrations (25, 50, 70, and 90%) of theextraction solvent (ethanol) in the mouse macrophage cell line treatedwith LPS, in order to investigate the anti-inflammatory effect and theanti-oxidative effect according to the extraction method (hot-waterextraction) of the complex herbal medicine extract and theconcentrations of the extraction solvent (ethanol) of the presentdisclosure.

FIGS. 11A, 11B and 11C are graphs comparing the expression levels of theinflammatory factors (IL-113, IL-6, and TNF-α) according to thetreatment with the single extracts of herbal medicines (Dioscoreaerhizoma, Taraxaci herba, and Schizonepetae spica) and the complex herbalmedicine extract in the pulmonary inflammation mouse models induced byLPS, in order to investigate the pulmonary inflammation inhibitoryeffect of the single extracts and complex extract of herbal medicines.

DETAILED DESCRIPTION

Hereinafter, the present disclosure will be described in more detailwith reference to examples. These examples are only for illustrating thepresent disclosure more specifically, and it would be apparent to thoseskilled in the art that the scope of the present disclosure is notlimited by these examples according to the gist of the presentdisclosure.

EXAMPLES

Throughout the present specification, the “%” used to express theconcentration of a specific material, unless otherwise particularlystated, refers to (wt/wt) % for solid/solid, (wt/vol) % forsolid/liquid, and (vol/vol) % for liquid/liquid.

Preparative Examples Preparative Example 1: Preparation of ComplexHerbal Medicine Extract (Mixed Extract)

Washed and dried Dioscoreae rhizoma, Taraxaci herba, and Schizonepetaespica were used in tests. The herbal medicines Dioscoreae rhizoma,Taraxaci herba, and Schizonepetae spica were mixed at a weight ratio of1:1:1 to a total of 60 g, and 25% (v/v) ethanol aqueous solution havinga volume 10 times the weight was added thereto, followed by extractionwhile well stirring at room temperature for 8 hours. The extract wasfiltered, concentrated under reduced pressure at 50-65° C., and thenfreeze-dried, thereby obtaining a powder-type complex herbal medicineextract (mixed extract). The yield was about 12-13%.

Preparative Example 2: Preparation of Complex Herbal Medicine Extract(Mixed Extract) According to Mixing Ratio of Herbal Medicines

Washed and dried Dioscoreae rhizoma, Taraxaci herba, and Schizonepetaespica were used in tests. The herbal medicines Dioscoreae rhizoma,Taraxaci herba, and Schizonepetae spica were mixed at weight ratiosshown in Table 1 to a total of 30 g, and 25% (v/v) ethanol aqueoussolution having a volume 10 times the weight was added thereto, followedby extraction while well stirring at room temperature for 8 hours. Eachof the extracts was filtered, concentrated under reduced pressure at50-65° C., and then freeze-dried, thereby obtaining a total of seventypes of complex herbal medicine extract powders. The yields thereof areshown in Table 1.

TABLE 1 Dioscoreae Taraxaci Schizonepetae Yield Classification RhizomaHerba Spica (%) Preparative Example 2-1 1 1 1 11.95 Preparative Example2-2 2 1 1 13.17 Preparative Example 2-3 4 1 1 14.81 Preparative Example2-4 1 2 1 11.84 Preparative Example 2-5 1 4 1 10.83 Preparative Example2-6 1 1 2 10.49 Preparative Example 2-7 1 1 4 10.27

Preparative Example 3: Preparation of Complex Herbal Medicine Extracts(Mixed Extracts) According to Various Concentrations of ExtractionSolvent (Ethanol)

Washed and dried Dioscoreae rhizoma, Taraxaci herba, and Schizonepetaespica were used in tests. The herbal medicines Dioscoreae rhizoma,Taraxaci herba, and Schizonepetae spica were mixed at the weight ratio(w/w) in Preparative Example 2-1 to a total of 30 g, and 25, 50, 70, and90% ethanol aqueous solutions having a volume 10 times the weight wereadded thereto, followed by extraction while well stirring at roomtemperature for 8 hours. Each of the extracts was filtered, concentratedunder reduced pressure at 50-65° C., and then freeze-dried, therebyobtaining a total of four types of complex herbal medicine extractpowders. The yields thereof are shown in Table 2.

TABLE 2 Ethanol aqueous solution Yield Classification concentration (%)(%) Note Preparative 25 12.37 Same preparation method Example 3-1 as inPreparative Example 2-1 Preparative 50 13.71 — Example 3-2 Preparative70  9.84 — Example 3-3 Preparative 90  5.23 — Example 3-4

Preparative Example 4: Preparation of Complex Herbal Medicine Extract(Mixed Extract) Through Hot-Water Extraction

Washed and dried Dioscoreae rhizoma, Taraxaci herba, and Schizonepetaespica were used in tests. The herbal medicines comprising Dioscoreaerhizoma, Taraxaci herba, and Schizonepetae spica were mixed at theweight ratio (w/w) in Preparative Example 2-1 to a total of 30 g, anddistilled water having a volume 10 times the weight was added thereto,followed by reflow extraction at a temperature of 90° C. for 3 hours.The extract was filtered, concentrated under reduced pressure at 50-65°C., and then freeze-dried, thereby obtaining a complex herbal medicineextract powder, and the yield thereof was about 13.30%.

Comparative Example 1: Preparation of Single-Herbal Medicine Extracts

Washed and dried Dioscoreae rhizoma, Taraxaci herba, and Schizonepetaespica were used in tests. To 30 g of each of the herbal medicinesDioscoreae rhizoma, Taraxaci herba, and Schizonepetae spica, 25% (v/v)ethanol aqueous solution having a volume 10 times the weight was added,followed by extraction while well stirring at room temperature for 8hours. Each of the extracts was filtered, concentrated under reducedpressure at 50-65° C., and then freeze-dried, thereby obtaining a totalof three types of single-herbal medicine extract powders. The yieldsthereof are shown in Table 3.

TABLE 3 Classification Kind of herbal medicine Yield (%) ComparativeExample 1-1 Dioscoreae Rhizoma 19.86 Comparative Example 1-2 TaraxaciHerba 12.52 Comparative Example 1-3 Schizonepetae Spica  7.84

Test Examples Test Example 1: Lung Tissue Damage Inhibitory Effect ofComplex Herbal Medicine Extract in PPE-Induced Emphysema Mouse Models

In order to investigate a lung tissue damage inhibitory effect of thecomplex herbal medicine extract of the present disclosure prepared inPreparative Example 1, the following test was carried out.

After 7-week-old male C57BL/6 mice (Raonbio, Korea) were acclimated forat least one week, the animals were classified into (1) a normal group,(2) a group with emphysema induction and complex herbal medicine extractadministration (test group), and (3) a group with emphysema inductionand distilled water administration (negative control group). For theinduction of emphysema in the test group and the negative control group,1 U of porcine pancreatic elastase (PPE, Millipore, USA) wasadministered as a single drop into the mouse trachea. For the normalgroup, phosphate buffered saline (PBS) was administered as a single dropinto the mouse trachea.

For the test group, the complex herbal medicine extract dissolved indistilled water was orally administered once/day at a dose of 200 mg/kgfor two weeks from one week before PPE administration. For the normalgroup and the negative control group, only distilled water was orallyadministered. After the last administration of the complex herbalmedicine extract or distilled water, the mice were anesthetized withcarbon dioxide and the lung tissue was extracted. The extracted lungtissue was fixed in formalin and subjected to hematoxylin and eosinstaining (H&E staining). The images taken by observing the lung tissuewith an optical microscope (×100) are shown in FIG. 1 .

As shown in FIG. 1 , as a result of inducing emphysema by PPE, thealveolar dilation and lung tissue damage in the negative control groupsignificantly increased by about 2.2 times compared with the normalgroup. In addition, the lung tissue damage was inhibited in the testgroup with administration of the complex herbal medicine extractcompared with the emphysema induction group (negative control group).

It could be therefore verified that the complex herbal medicine extractof the present disclosure showed a lung tissue damage inhibitory effectin the emphysema mouse models.

Test Example 2: Inhibitory Effect of Complex Herbal Medicine Extract onFine Dust-Induced Reactive Oxygen Species Formation of PulmonaryEpithelial Cell Line

In order to investigate a reactive oxygen species formation inhibitoryeffect of the complex herbal medicine extract of the present disclosureprepared in Preparative Example 1, the following test was carried out.

The human pulmonary endothelial cell line NCI-H292 cells (ATCC, USA)were incubated in an incubator of 5% CO₂ and 37° C. by using RPMI media(Corning, USA) comprising 10% fetal bovine serum (FBS). The cells wereprepared on a 96-well plate at 1×10⁴ cells per well, and stabilized for24 hours. After the cells were stabilized, the cell supernatant wasremoved, and then the cells were treated with the complex herbalmedicine extract in Preparative Example 1 at 100 μg/ml, followed byincubation for 1 hour. Then, the cells were further treated with 200μg/ml fine dust (particulate matter 10, PM10, NIST, USA) (test group).The normal group was treated with neither the complex herbal medicineextract nor fine dust, and the negative control group was treated withonly fine dust. After 3 hours, the amount of reactive oxygen speciesformed in the cells was investigated by using DCF-DA analysis (SigmaAldrich, USA).

As shown in FIG. 2 , the reactive oxygen species was increased by about125% in the group with fine dust induction (negative control group)compared with the normal group, and such reactive oxygen species wasinhibited by about 61% through the complex herbal medicine extract. Itcan be therefore seen that the complex herbal medicine extract of thepresent disclosure showed an excellent antioxidative effect.

Test Example 3: Inhibitory Effect of Complex Herbal Medicine Extract onFine Dust-Induced Apoptosis

As in Test Example 2, NCI-H292 cells were prepared on a 96-well plate at1×10⁴ cells per well, and stabilized. After 24 hours, the cellsupernatant was removed, and then the cells were treated with thecomplex herbal medicine extract in Preparative Example 1 atconcentrations of 100, 200, 400, and 800 mg/mL. After 1 hour, the cellswere further treated with 50 μg/mL fine dust (PM 10). The normal groupwas treated with neither the complex herbal medicine extract nor finedust, and the negative control group was treated with only fine dust.After 24 hours, cell viability was investigated through the WST-1 test(Younginfrontier, Korea).

As shown in FIG. 3 , as a result of treating the pulmonary epithelialcell line with fine dust, cell viability was about 63.6% compared withthe normal group by apoptosis (negative control group). Whereas, thetest groups with the treatment of the complex herbal medicine extract atconcentrations of 100, 200, 400, and 800 μg/mL showed cell viability of67.5%, 69.9%, 77.2%, and 89%, respectively, indicating that apoptosiswas inhibited in a dose-dependent manner. It can be therefore seen thatthe complex herbal medicine extract of the present disclosure showed anexcellent apoptosis inhibitory effect.

Test Example 4: Anti-Aging Effect of Complex Herbal Medicine Extract onFine Dust-Induced Cell Aging of Pulmonary Epithelial Cell Line

As in Test Example 2, NCI-H292 cells were prepared on a 96-well plate at1×10⁴ cells per well, and stabilized. After 24 hours, the cellsupernatant was removed, and then the cells were treated with thecomplex herbal medicine extract in Preparative Example 1 at aconcentration of 200 μg/ml. After 1 hour, the cells were further treatedwith 50 μg/mL fine dust (PM 10). The normal group was treated withneither the complex herbal medicine extract nor fine dust, and thenegative control group was treated with only fine dust. After five days,the cells were fixed in formalin, and then the degree of cell aging wasinvestigated through senescence beta-galactosidase staining.

As shown in FIG. 4 , cell aging caused by fine dust treatment wassignificantly reduced in the test group treated with the complex herbalmedicine extract compared with the negative control group. It can betherefore seen that the complex herbal medicine extract of the presentdisclosure showed an excellent anti-aging effect.

Test Example 5: Telomerase Expression Increasing Effect of ComplexHerbal Medicine Extract in Pulmonary Endothelial Cell Line (Anti-AgingEffect)

NCI-H292 cells were prepared on a 100 pi (φ) plate at 1×10⁶ cells andstabilized. After 24 hours, the cells were treated with the complexherbal medicine extract in Preparative Example 1 at concentrations of100 and 200 μg/ml. After 24 hours, the cells were collected and proteinswere extracted, and then western blotting was performed using anantibody (ab32020, Abcam, USA) for human telomerase reversetranscriptase (hTERT), which is a catalytic small unit of telomerase.

As shown in FIG. 5 , the pulmonary endothelial cell line was treatedwith the complex herbal medicine extract at concentrations of 100 and200 μg/ml, and as a result, the expression of hTERT dose-dependentlyincreased to 1.6 times and 2.2 times, respectively, compared with thenon-treatment group (control group). It can be therefore seen that thecomplex herbal medicine extract of the present disclosure showed anexcellent anti-aging effect.

Test Example 6: Inhibitory Effect (Anti-Inflammatory Effect) of ComplexHerbal Medicine Extract on LPS-Induced Nitric Oxide (NO) Production inMacrophage Cell Line

The mouse macrophage cell line Raw 264.7 cells (ATCC, USA) wereincubated in the incubator of 5% CO₂ and 37° C. by using RPMI media(Invitrogen, USA) comprising 10% fetal bovine serum (FBS). The cellswere prepared on a 24-well plate at 2.5×10⁵ cells per well, andstabilized. After 24 hours, the cell supernatant was removed, and thenthe cells were treated with the complex herbal medicine extract inPreparative Example 1 at concentrations of 0.5, 1, 2, and 4 mg/mL. After1 hour, the cells were further treated with 100 ng/mL LPS. After 24hours, the cell supernatant was collected and subjected to Griess testfor measuring the change in NO production, and the concentration of NOwas calculated using the standard curve according to the concentrationof sodium nitrite (NaNO₂) (FIG. 6 ).

As shown in FIG. 6 , the production of NO, which is an inflammatoryfactor, increased to a level of 12 μM by LPS treatment in macrophages(negative control group). In the test group treated with the complexherbal medicine extract at concentrations of 0.5, 1, 2, and 4 mg/mltogether with LPS, the NO concentrations were 13.6, 9.4, 3.6, and 3 μM,respectively, showing a concentration-dependent reduction. It can betherefore seen that the complex herbal medicine extract of the presentdisclosure showed an excellent anti-inflammatory effect.

Test Example 7: Inhibitory Effect (Anti-Inflammatory Effect) of ComplexHerbal Medicine Extract on LPS-Induced Inflammatory Factor Expression inMacrophage Cell Line

The mouse macrophage cell line Raw 264.7 cells (ATCC, USA) wereincubated in an incubator of 5% CO₂ and 37° C. by using RPMI media(Invitrogen, USA) comprising 10% fetal bovine serum (FBS). The cellswere prepared on a 24-well plate at 2.5×10⁵ cells per well, andstabilized. After 24 hours, the cell supernatant was removed, and thenthe cells were treated with 100 ng/mL LPS (Sigma, US) and the complexherbal medicine extract in Preparative Example 1 at concentrations of0.5, 1, and 2 mg/mL. After 24 hours, the cell supernatant was removed,and then RNA was separated from the cells by using TRIzol (Invitrogen,USA). Thereafter, cDNA obtained through RT-PCR was used to perform qPCRusing primers specific to the inflammatory factors IL-6, IL-1β, and iNOSand the SYBR green probe (Takara, Japan). The RNA expression changevalue obtained from qPCR was expressed as a relative change of GAPDHmRNA as a standard gene compared with a non-treatment group (FIGS. 7A to7C). The primer sequences for mouse genes used in the test are shown inTable 4.

TABLE 4   SEQ Nucleotide Sequence ID Gene Direction (5′ to 3′) NO GAPDHForward AGCCTCGTCCCGTAGACAA 1 Reverse AATCTCCACTTTGCCACTGC 2 IL-6Forward TTGGTCCTTAGCCACTCCTTC 3 Reverse TAGTCCTTCCTACCCCAATTTCC 4 IL-1β Forward TGTGCAAGTGTCTGAAGCAGC 5 Reverse TGGAAGCAGCCCTTCATCTT 6 iNOSForward CGAAACGCTTCACTTCCAA 7 Reverse TGAGCCTATATTGCTGTGGCT 8

As shown in FIGS. 7A to 7C, the production of the inflammation factorsIL-6, IL-1β, and iNOS significantly increased in the Raw 264.7macrophages by LPS treatment (negative control group), and in the testgroup treated with the complex extract of three kinds of herbalmedicines in Preparative Example 1 at concentrations of 0.5, 1, and 2mg/ml together with LPS, all the expression levels of IL-6, IL-1β, andiNOS were significantly reduced in a dose-dependent manner. It can betherefore seen that the complex extract of three kinds of herbalmedicines of the present disclosure showed an excellentanti-inflammatory effect.

Test Example 8: Antioxidative Factor Expression Increasing Effect(Antioxidative Effect) of Complex Herbal Medicine Extract in MacrophageCell Line Treated with LPS

In order to investigate the effect of the complex herbal medicineextract of the present disclosure on the expression of the antioxidativefactor heme oxygenase-1 (HO-1) in the macrophage cell line induced byLPS, the test was carried out by the same method as in Test Example 7except that qPCR was performed by using primers specific to HO-1 geneand SYBR green probe (Takara, Japan). The RNA expression change valueobtained from the qPCR results was expressed as a relative change ofGAPDH mRNA as a standard gene compared with a non-treatment group (FIG.8 ). The primer sequences for mouse genes used in the test are shown inTable 5.

TABLE 5   SEQ Nucleotide Sequence ID Gene Direction (5′ to 3′) NO GAPDHForward AGCCTCGTCCCGTAGACAA  1 Reverse AATCTCCACTTTGCCACTGC  2 HO-1Forward CAGGTGATGCTGACAGAGGA  9 Reverse GAGAGTGAGGACCCACTGGA 10

As shown in FIG. 8 , in the test group treated with the complex herbalmedicine extract in Preparative Example 1 at concentrations of 0.5, 1,and 2 mg/ml together with LPS, the expression level of HO-1significantly increased in a dose-dependent manner. It can be thereforeseen that the complex herbal medicine extract of the present disclosureshowed an excellent anti-oxidative effect.

Test Example 9: Antioxidative Factor Expression Increasing Effect(Antioxidative Effect) of Complex Herbal Medicine Extract According toMixing Ratio of Herbal Medicines in Macrophage Cell Line Treated withLPS

In order to investigate an antioxidative effect of the complex herbalmedicine extract in Preparative Example 2 according to the mixing ratioin the Raw 264.7 macrophage cell line induced by LPS, qPCR was performedby using the primers specific to HO-1 gene and the SYBR green probe(Takara, Japan). The detailed test procedure was the same as in TestExample 8, and the primer sequences for the mouse genes used in the testare shown in Table 5 above.

As shown in FIG. 9 , all the test groups treated with the complex herbalmedicine extracts in Preparative Example 2 at a concentration of 2 mg/mltogether with LPS showed an excellent antioxidative effect bysignificantly increasing the expression level of the antioxidativefactor HO-1.

Test Example 10: Anti-Inflammatory and Antioxidative Effects of ComplexHerbal Medicine Extract According to Concentration of Extraction Solvent(Ethanol) in Macrophage Cell Line Treated with LPS

In order to investigate the anti-inflammatory and antioxidative effectsof the complex herbal medicine extracts according to the concentrationsof the extraction solvent (ethanol) in Preparative Example 3 and thehot-water complex herbal medicine extract in Preparative Example 4, qPCRwas performed by using the primers specific to iNOS, and HO-1 and theSYBR green probe (Takara, Japan). Each of the extracts was used at aconcentration of 2 mg/ml. The detailed test procedures were the same asin Test Examples 7 and 8, and the primer sequences for the mouse genesused in the tests are shown in Tables 4 and 5 above.

As shown in FIG. 10 , the complex herbal medicine extracts according tothe concentrations of the extraction solvent (ethanol) prepared inPreparative Example 3 significantly reduced the expression of iNOSincreased by LPS treatment, showing an excellent anti-inflammatoryeffect. In addition, all the complex herbal medicine extracts inPreparative Examples 3 and 4 significantly increased the expressionlevel of the antioxidative factor HO-1, showing an excellentantioxidative effect.

Test Example 11: Anti-Inflammatory Effects of Single Herbal MedicineExtract and Complex Herbal Medicine Extract in Pulmonary InflammationMouse Model Induced by LPS

In order to investigate the pulmonary inflammation inhibitory effect ofsingle and complex herbal medicine extracts, the following test wascarried out. After 7-week-old male C57BL/6 mice (Raonbio, Korea) wereacclimated for at least one week, the animals were classified into (1) anormal group, (2) a group with LPS induction and distilled wateradministration (negative control group), (3) to (5) groups with LPSinduction and single herbal medicine extract administration, and (6) agroup with LPS induction and complex herbal medicine extractadministration.

The complex herbal medicine extract in Preparative Example 1 dissolvedin distilled water and the single extracts of three kinds of herbalmedicines in Comparative Example 1 dissolved in distilled water wereorally administered to the test groups of (3) to (6) at 500 mg/kgonce/day for five days, and only distilled water was orally administeredto the normal group and the negative control group.

The induction of acute pulmonary inflammation in the test groups and thenegative control group was performed by administering 50 μg of LPS(Sigma, US) dissolved in 50 μl of phosphate buffered saline (PBS) as asingle drop into the mouse trachea 24 hours before the end of the test.For the normal group, phosphate buffered saline (PBS) was administeredas a single drop into the mouse trachea.

After the last administration of the herbal medicine extracts ordistilled water, the mice were sacrificed with carbon dioxide, and thenthe lung tissue was separated, and RNA was extracted by using TRIzol(Invitrogen, USA). Thereafter, cDNA obtained through RT-PCR was used toperform qPCR using primers specific to the inflammatory factors IL-1β,IL-6, and TNF-α and the SYBR green probe (Takara, Japan). The RNAexpression change value obtained from qPCR was expressed as a relativechange of GAPDH mRNA as a standard gene compared with a non-treatmentgroup. The primer sequences for mouse genes used in the test are shownin Table 6.

TABLE 6   SEQ Nucleotide Sequence ID Gene Direction (5′ to 3′) NO GAPDHForward AGCCTCGTCCCGTAGACAA  1 Reverse AATCTCCACTTTGCCACTGC  2 IL-6Forward TTGGTCCTTAGCCACTCCTTC  3 Reverse TAGTCCTTCCTACCCCAATTTCC  4IL-1β Forward TGTGCAAGTGTCTGAAGCAGC  5 Reverse TGGAAGCAGCCCTTCATCTT  6TNF-α Forward AAGCCTGTAGCCCACGTCGTA 11 Reverse GGCACCACTAGTTGGTTGTCTTTG12

As shown in FIGS. 11A to 11C, the expression of the inflammatory factorsIL-1β, IL-6, and TNF-α within the lung tissue was increased by LPS. Theadministration of each of the single extracts of three kinds of herbalmedicines showed expression levels of the inflammatory factors similarto those in the negative control group. Whereas, the complex herbalmedicine extract reduced the expression of the inflammatory factors tosignificant levels compared with the negative control group, therebyshowing a synergistic effect in the anti-inflammatory effect.

1.-6. (canceled)
 7. A oral composition comprising a mixed extract ofDioscoreae rhizoma, Taraxaci herba, and Schizonepetae spica as an activeingredient, wherein a weight ratio of the Dioscoreae rhizoma, Taraxaciherba, and Schizonepetae spica in the mixed extract is 1-4:1:1-4 or1:1-4:1-4, wherein the extract is obtained by extraction with C1 to C6lower alcohol or aqueous solution thereof, and wherein the oralcomposition is selected from the group consisting of an emulsion,granules, a tablet, and a capsule.
 8. The oral composition of claim 7,wherein the C1 to C6 lower alcohol is ethanol.
 9. The oral compositionof claim 7, the mixed extract is an extract from a mixture of Dioscoreaerhizoma, Taraxaci herba, and Schizonepetae spica.
 10. The oralcomposition of claim 7, the mixed extract is a mixture of Dioscoreaerhizoma extract, Taraxaci herba extract, and Schizonepetae spicaextract.
 11. A food composition comprising (a) a mixed extract ofDioscoreae rhizoma, Taraxaci herba, and Schizonepetae spica, and (b) aningredient selected from the group consisting of a protein, acarbohydrate, a fat, a nutrient, a seasoning, and a flavoring agent,wherein the Dioscoreae rhizoma, Taraxaci herba, and Schizonepetae spicaare in a weight ratio of 1-4:1:1-4 or 1:1-4:1-4, and wherein the extractis obtained by extraction with C1 to C6 lower alcohol or aqueoussolution thereof.
 12. The food composition of claim 11, wherein the foodis health functional food selected from the group consisting of drinks,meats, chocolates, foods, confectionery, pizzas, instant noodles, gums,ice creams, alcohol drinks, vitamin complexes, and health supplementfoods.
 13. A method for alleviation or treatment of a respiratorydisease comprising: administering a composition to a subject in needthereof, the composition comprising a mixed extract of Dioscoreaerhizoma, Taraxaci herba, and Schizonepetae spica as an activeingredient, wherein a weight ratio of the Dioscoreae rhizoma, Taraxaciherba, and Schizonepetae spica in the mixed extract is 1-4:1:1-4 or1:1-4:1-4, and wherein the extract is obtained by extraction with C1 toC6 lower alcohol or aqueous solutions thereof.
 14. The method of claim15, wherein the respiratory disease is selected from the groupconsisting of rhinitis, pharyngitis, laryngitis, pharyngolaryngitis,pneumonia, acute or chronic bronchitis, and chronic obstructivepulmonary disease.
 15. The method of claim 15, wherein the C1 to C6lower alcohol is ethanol.